Self-Assembly of Synthetic and Biological Polymeric Systems of ...

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r h,app = kT/(6πηD app ) (1)
where k is the Boltzmann constant and η is the viscosity of water
at temperature T.
Thioflavin T spectroscopy.
Protein and ThT were dissolved in buffer at a final proteindye
molar ratio of 50:1. Samples were continuously stirred only
during data acquisition intervals in order to avoid ThT deposition
onto protein aggregates/fibrils. Fluorescence was measured in a
Cary Eclipse fluorescence spectrophotometer equipped with a
temperature controller and a multi-cell sample holder (Varian
Instruments Inc.). Excitation and emission were at wavelengths of
450 and 482 nm, respectively. All intensities were backgroundcorrected
for the ThT-fluorescence in the respective solvent in the
absence and presence of monomeric HSA at pH 7.4 and 2.0.
Circular Dichroism (CD).
Far-UV circular dichroism (CD) spectra were obtained using
a JASCO-715 automatic recording spectropolarimeter with a
JASCO PTC-343 Peltier-type thermostated cell holder. Quartz
cuvettes with 0.2 cm pathlength were used. The protein
concentration was reduced to 0.1 mg/mL in order to avoid light
scattering effects as much as possible due to protein aggregation.
CD spectra were obtained from aliquots withdrawn from the
aggregation mixtures at the indicated conditions after incubation,
and recorded at wavelengths between 195 and 300 nm at 25 ºC.
The mean residue ellipticity θ (deg cm 2 dmol -1 30
) was calculated
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according to: θ=(θObs/10)/(MRM/lc), where θobs is the
observed ellipticity (in deg), MRM is the mean residue molecular
mass (in g mol -1 ), l is the optical path-length (in cm), and c is the
protein concentration (in g ml -1 ). The secondary structure of the
protein was calculated from far-UV CD spectra by running the
SELCON3, CONTIN, and DSST programs within the
DICHROWEB server. 45,46 Final results were assumed when data
generated from all programs showed convergence.
Transmission electron microscopy (TEM).
HSA solutions were applied to carbon-coated copper grids,
blotted, washed, negatively stained with 2% (w/v) of
phosphotungstic acid, air dried, and then examined with a Phillips
CM-12 transmission electron microscope operating at an
accelerating voltage of 120 kV. Samples were diluted between
20-200-fold prior deposition onto the grids.
Results and Discussion
To induce HSA aggregation and subsequent amyloid
formation we use 10 mg/mL HSA solutions at either pH 7.4 or
pH 2.0 and ionic strength of 10 mM. At pH 7.4 the protein is in
its native state whereas at pH 2.0 it is in its E-expanded one,
which is characterized by a separation of domain III and a loss of
the intradomain helices of domain I. 47 Monomer sizes and
structural compositions were similar to those previously
reported. 40 Samples were prepared by adding specified volumes
of protein stock solutions and different amounts of ethanol, which
has been shown to destabilize both the secondary and tertiary
structures of HSA; 36 subsequently, samples were incubated at
room or elevated temperature (65 ºC, above the melting
temperature of HSA) 48,49 to induce further protein structural
changes and aggregation.
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Aggregate formation.
The formation of HSA aggregates and their aggregation
kinetics were firstly analyzed by SLS. Figure 1 shows the time
evolution of the scattered light intensity of HSA samples at
varying pH, temperature and alcohol content conditions upon
incubation. Different curve profiles are obtained depending on
incubation conditions.
At physiological pH (Figure 1a-b), the plots reflect a quick
increase in the scattered intensity at short incubation times, which
denotes the fast formation of protein aggregates. SLS plot profiles
suggest a mechanism of continuous protein aggregation (in
particular, a continuous fibrillation process as later confirmed by
ThT, CD and TEM data) without a nucleation step, as denoted the
absence of a lag phase in the early periods of incubation (see inset
in Figure 1a). In addition, the absence of a faster aggregation
process when protein seeds (i.e. protein aggregates) are added to
protein solutions followed by subsequent incubation, and the
decrease in the aggregation rate if the starting protein
concentration is lowered (see Figure S1 in ESI) further indicate
that under the present conditions HSA aggregation occurs by
means of a classical coagulation or downhill polymerization
mechanism, i.e. each protein monomer association is bimolecular,
effectively irreversible and thermodynamically favorable, so that
there is no energy barrier to impede aggregate growth. 50 A similar
behavior has been previously obtained in the absence of
alcohol. 40-42,50,51 ThT fluorescence data confirmed similar trends
as those observed by SLS (see Figure S2). High values of ThT
fluorescence point to an important formation/gain of β-sheet
structure along the protein aggregation process, which is a first
evidence of the possible formation of HSA fibrils during the
aggregation process, 37 as further confirmed below by CD and
TEM techniques.
Also, under physiological conditions we observe that as the
ethanol concentration increases up to 60% (v/v) the formation of
more numerous and/or bulkier protein aggregates takes place as
derived from both the increments in scattered intensities –
proportional to protein concentration and molecular weight
(Figure 1a,b), and the shifts of the aggregate size distributions to
larger values (see Figure 2a-b below). The clustering of alcohol
molecules and the enhancement of alcohol-water interactions at
intermediate ethanol concentrations in the mixed solvent should
modify the extent of protein hydration and molecular packing 53 as
confirmed by CD and FT-IR below, favoring an enhanced protein
aggregation. At larger alcohol concentrations (> 60% (v/v)),
protein solutions became progressively turbid as a consequence
of the formation of even larger aggregates, 52 which gradually
precipitated (see below for further explanation).